The male germ unit association is independently regulated of GUM in Arabidopsis thaliana

Abstract Cytoplasmic projections (CPs) formed by the generative and sperm cells link the male gametes with the vegetative cell (VC) nucleus, which are required to build the male germ unit (MGU) assemblage in the angiosperm pollen grain. As molecular and genetic controls underlying CP development and formation of the MGU are unknown, it was hypothesized that physical association between germ cells and the VC nucleus might be lost in germ unit malformed (gum) mutants or in those which either block generative cell (GC) division or that additionally prevent gamete differentiation. In vivo, analysis of marked cellular components demonstrated a linkage of sperm cells (SCs) and the VC nucleus in gum mutant alleles despite their increased physical separation. Similarly, for several independent classes of bicellular pollen mutants, undivided GCs were associated with the VC nucleus like GCs in wild‐type pollen. We conclude that the early formation of GC CPs to establish the MGU is regulated independently of DUO1‐DAZ1 and DUO3 transcription factors as well as cyclin‐dependent kinase function (CDKA;1). As the absence of cytoplasmic protrusion was expected in the gum mutants in Arabidopsis, early histological studies reported temporal disappearance of cytoplasmic protrusion in several organisms. Our findings demonstrated the striking importance of live imaging to verify the broad conservation of the persistent MGU contact in all the angiosperms and its important role in successful double fertilization.


| INTRODUCTION
The male germline cells (generative cell [GC]/sperm cell [SC]) in developing pollen of angiosperms form fine cytoplasmic projections (CPs) which physically link the male gametes with the pollen vegetative cell (VC) nucleus to create a structural assemblage, known as the male germ unit (MGU) (Dumas et al., 1985).Evidence for the MGU was first observed in pollen of cotton (Jensen & Fisher, 1968), and one of the SCs of the pair is linked with the VC nucleus by a CP (Braun & Winkelmann, 2016;Goto et al., 2020;Kliwer & Dresselhaus, 2010;McConchie, Hough, & Knox, 1987;McCue et al., 2011;Zee & Ye, 2000;Zhou & Meier, 2014).The physical association between the GC and VC nucleus has been documented in early to mid-bicellular stage pollen (Table 1).This association is observed in the majority of bicellular pollen species, which shed bicellular pollen at anthesis and within the pollen tube (Table 1).Similarly, the MGU association has also been observed in many tricellular pollen species at different developmental stages (Table 2).The presence of the MGUs in angiosperm pollen and their presumptive importance in double fertilization is now largely accepted (reviewed in Mogensen, 1992;Dumas et al., 1998;McCue et al., 2011).
In Arabidopsis, the physical connection between the SC pair and VC nucleus to form the MGU has been documented previously in mature pollen via light, epifluorescence, live cell imaging of marker lines, and confocal laser scanning microscopy (CLSM) microscopic methods (Ge et al., 2011;Goto et al., 2020;Hamamura et al., 2011;Lalanne & Twell, 2002;Motomura et al., 2021;Reňák et al., 2012;Vogler et al., 2015;Xie et al., 2010;Zhou & Meier, 2014).However, there is limited information about the developmental dynamics of MGU assembly (Lalanne & Twell, 2002), and it remains unknown whether this association also exists at different bicellular pollen stages in Arabidopsis.The physical contact between the male germline cells and the vegetative nucleus (VN) via the CP establishes cell-to-cell communication, which might play an important role in the delivery of substances and/or transport of an intact MGU through the pollen tube for the process of double fertilization (McCue et al., 2011).
It was proposed that the physical association of SC (SC1) with the VN facilitates the transfer of material or information between these two structures (Mogensen, 1992).Furthermore, the transport of substances may be directly from the VN to the SC through CP (as there is a membrane continuity between the vegetative nuclear envelope and plasma membrane surrounding GC/SC) or indirectly from the VC cytoplasm via membrane transport.The nuclear pore density is much greater where the GC/SC is associated with the VC nucleus in Medicago sativa (Shi et al., 1991).The MGU association between SC and VN provides an increased surface area for transport and interaction (Yu et al., 1992).Therefore, the increased surface area will need more energy for trafficking at the interface, and it has been reported that high ATPase hydrolysis occurs in the pollen of Hippeastrum vitatum and Clivia miniata (Pei-hua, 1988).The unidirectional transfer from the VC nucleus to SCs is suggested based on findings that an artificial microRNA expressed from the VC nucleus can reduce the expression of a target green fluorescent protein (GFP) reporter transcript in Arabidopsis SCs (Slotkin et al., 2009), while it has been reviewed that microRNA acts autonomously in plant body (Voinnet, 2005).The molecular mechanism has also been determined where the VC regulates transcriptional reactivation of transposable elements (TEs) and those TE transcripts degrade into small interfering RNAs (siRNAs), which resulted in silencing in SCs (Martinez et al., 2016).The movement of MGU in the pollen tube is regulated by the presence of WIPs (WPP domain-interacting proteins) and WITs (WPP domain-interacting tail-anchored proteins) proteins (Zhou & Meier, 2014), while the nuclear laminal protein, KAKU4 deficiency transforms the folded VN to a spherical form, which has resulted in low fertilization in A. thaliana (Goto et al., 2020).
There are convincing pieces of evidence about the MGU as a structural unit, and growing indication for its role as a conduit for cell-to-cell communication; however, the genetic and molecular mechanisms of this association remain largely obscure, which has been previously claimed to be regulated by GUM (Lalanne & Twell, 2002).Here, we describe the development of the MGU from its inception in early bicellular pollen and examine its dynamic organization in developing pollen of wild-type Arabidopsis as well as in several independent regulatory classes of bicellular pollen mutants.
In addition to these mutants, the allelic germ unit malformed (gum1-1/gum1-2) mutants were revisited that were known to regulate the MGU association (Lalanne & Twell, 2002) by determining the presence of MGU through the introduction of double fluorescent markers.
Therefore, we decided to mark the male germline (GC/SC) and vegetative nuclear membrane with fluorescent markers for the cellular morphology and confirm the presence of MGU association through CP.The MGU mutants (i.e., gum/mud) were identified in the EMS mutagenized wild-type A. thaliana pollen (Lalanne & Twell, 2002).In the mud (MGU displaced) mutant, the intact MGU is displaced toward the pollen grain wall, while in the gum mutant, the physical contact between the SC and VN (VN) appears to be lost at late tricellular pollen stages.It was proposed that MUD potentially regulates the position of the MGU and GUM encode proteins to keep the SC and VN linked and/or close to each other (Lalanne & Twell, 2002).
The duo1 and duo2 mutants were found in an EMS-mutagenized DAPI screen in Arabidopsis pollen, where the duo2 mutant GC enters mitosis but arrests at prometaphase, highlighting the role of DUO2 in mitotic progression and mutant GC in duo1 completes the S-phase but fails to enter mitosis (Durbarry et al., 2005).DUO POLLEN 1 (DUO1) was the first transcription factor shown to be expressed specifically in the GCs (Rotman et al., 2005), and its transcript level peaks during male gametogenesis before GC division (Brownfield et al., 2009).Similarly, the GC division is regulated by DUO2 independently of DUO1, based on the HTR10 (DUO1 target) expression in the duo2 mutant germline (Figure S12) as the gene has not been characterized and the molecular mechanism of mutation is unknown.
The DUO POLLEN 3 (DUO3) regulates GC division because mutant GC enters the S-phase but fails to divide, while coordinates with DUO1 for the expression of GEX2 and GCS1 (HAP2) in the germline (Brownfield et al., 2009;Brownfield & Twell, 2009).Furthermore, it has been proposed that both DUO1 and DUO3 activate an T A B L E 1 Review of evidence for male germ unit association among bicellular pollen species.The male germ unit has been documented in 49 out of 55 bicellular pollen species studied at the developmental stages indicated.The male germ unit was not detected only in two studies highlighted in gray (study IDs.53 and 54) of Helleborus foetidus (Heslop-Harrison et al., 1986) and Haemanthus katherinae (Sanger & Jackson, 1971)   T A B L E 2 Review of evidence for male germ unit association among tricellular pollen species.The male germ unit has been documented in all 46 tricellular pollen species studied at the developmental stages indicated.The male germ unit was not detected in nine studies (study IDs. 6,25,(27)(28)30,33,(36)(37)40) that included those on Arabidopsis thaliana (Derksen et al., 2002;Kuang & Musgrave, 1996;Owen & Makaroff, 1995;Yamamoto et al., 2003), Zea mays (Rusche & Mogensen, 1988), and Hordeum vulgare (Mogensen & Rusche, 1985), but independent studies have subsequently confirmed the presence of the male germ unit in all three species (Hamamura et al., 2011;Kliwer & Dresselhaus, 2010;Mogensen & Wagner, 1987;Reňák et al., 2012).

Species
which have an important role in the G2-M transition of the GC division and mitotic cyclins accumulation (Borg et al., 2014).
It has been shown that the F-box like-17 protein forms a complex with SKP1-like protein 11 (ASK11), which degrades the Kip-related protein 6 (KRP6) and KRP7 in the male germline and regulates GC progress through PMII during cell cycle, while mutation of an F-box protein (FBL17) results in bicellular pollen mutant known as fbl17 (Kim et al., 2008;Gusti et al., 2009).The plant A-type cyclindependent kinase (CDKA) is a homolog of the animal cell division cycle protein 2 (CDC2) and plays an important role in the cell cycle.It has been demonstrated that CDKA;1 is required for GC entry into the cell cycle and both G 1 /S and G 2 /M phase transition are under its regulatory control, while loss-of-function mutation in CDKA;1 results in bicellular pollen containing a single sperm-like cell and a VC (Iwakawa et al., 2006).
To confirm the presence of physical contact between SC and VN in the gum allelic mutants ( gum1-1/gum1-2) and other bicellular pollen mutants (duo1, duo2, duo3, daz1daz2, cdka;1, fbl17), the double homozygous marker lines of mutants need to be examined developmentally.To achieve this, we introduced male germline (GC/SC) plasma membrane (TET11-GFP/DUO1:TET11-GFP), cytoplasm (HTR10:GFP-TUA6), and vegetative nuclear membrane (LAT52:Ran-GAP-tdTomato) fluorescent markers in the wildtype and different mutants.The application of these molecular tools was very helpful in marking the narrow CP and its association with the nuclear envelope.

| Plant materials and growth conditions
A. thaliana plants were grown in growth rooms on Levington F2S soil (Compost: Sand: Vermiculite, Scotts-UK) at 22 C with a 16-h-light and 8-h-dark cycle or with 24 h light (120 to 140 μ mol/m 2 /s) with 50%-60% relative humidity.

| Current status of the MGU association in angiosperms
The available published literature survey revealed the existence of MGU association in 49 out of 55 bicellular pollen species studied at the developmental stages (Table 1).The MGU was not detected only in Helleborus foetidus (Heslop-Harrison et al., 1986) and Haemanthus katherinae (Sanger & Jackson, 1971).Similarly, the MGU has been reported in all the 46 tricellular pollen species studied at the developmental stages, while only in nine studies (study IDs. 6,25,(27)(28)30,33,(36)(37)40) that included those on A. thaliana (Derksen et al., 2002;Kuang & Musgrave, 1996;Owen & Makaroff, 1995;Yamamoto et al., 2003), Zea mays (Rusche & Mogensen, 1988) and Hordeum vulgare (Mogensen & Rusche, 1985), but independent F I G U R E 1 Formation of the MGU in developing bicellular pollen of wild-type Arabidopsis.(a) Panels a-e show DIC and f-j the corresponding GFP signal from GFP-TUA6 (germline cytoplasm) images in early bicellular (a, f) to late bicellular (e, j) pollen marker lines (developmental analysis of eight-independent marker lines and more than 70 pollen grains examined at each independent bud stage).The generative cell initially has a round profile (a, f) and develops a fine thread-like cytoplasmic projection (g-j) that seems linked to the vegetative cell nucleus at all stages (b-e).
(b) Panels a-e show the GFP signal from GFP-TUA6 (germline cytoplasm) and f-j the corresponding RFP signal from RanGAP-tdTomato (vegetative cell nuclear envelope) in early bicellular (a, b) to late bicellular (e, j) pollen of double marker lines (six-independent homozygous marker lines analyzed, while the male germ unit association reported in 100% [90 pollen] pollen grains examined at each bud stage).The generative cell is round in profile (a, b) and then develops a fine thread-like cytoplasmic projection linked to the vegetative cell nuclear envelope at subsequent stages (c-e).Each image represents an independent pollen stage arranged from early to late pollen stages (left to right).Scale bar = 5 μm.
(c) Movies.Three-dimensional reconstruction of the male germ unit in wild-type at bicellular pollen stages.DIC, differential interference contrast; GFP, green fluorescent protein; MGU, male germ unit; RFP, red fluorescent protein.

| The MGU association at bicellular pollen stages
In the current in vivo developmental analysis, at the early bicellular pollen stage just after the detachment of the GC from the pollen wall, the GC remains close to the VC nucleus in the majority of pollen grains (Figure 1

| The MGU association at tricellular pollen stages
The GC divides symmetrically by pollen mitosis-II (PMII) into two SCs F I G U R E 5 Intact male germ unit in gum1-2 pollen at tricellular pollen stages.Panels a-e show DIC, f-j the GFP signal from TET11-GFP (germline plasma membrane), and k-o the corresponding RFP signal from RanGAP-tdTomato (vegetative cell nuclear envelope) in early tricellular (a, f, k) to late tricellular (e, j, o) pollen of a double marker line.One of the sperm cell pairs always appears linked to the vegetative cell nuclear envelope through its cytoplasmic projection even though the vegetative cell nucleus is located peripherally rather than centrally (lower panels, k-o) (five-independent homozygous marker lines analyzed developmentally for the presence of physical association in 100-140 pollen grains at different bud stages).Each image represents an independent pollen stage arranged from early to late bicellular pollen stages (left to right).Scale bar = 5 μm.GFP, green fluorescent protein; RFP, red fluorescent protein.
the SC and VN linked and/or close to each other (Figure 4) and a potential candidate gene involved in the MGU association (Lalanne & Twell, 2002).We did not present developmental data of mud (MGU displaced) mutant since the organization was not affected but instead, the whole MGU was displaced from the centre to the periphery (Lalanne & Twell, 2002).Therefore, in vivo, fluorescence microscopic analysis of gum mutants ( gum1-1/1-2) double homozygous marker lines, i.e., TET11-GFP Â DUO3:H2B-tdTomato and TET11-GFP Â LAT52:RanGAP-tdTomato, may help to elucidate the organization and assembly of the MGU as well as the molecular mechanisms.
The physical association between germline and vegetative nuclear envelope has been observed through CP at various bud stages in the five independent homozygous backed-crossed-6 (BC-6) gum1-2 (100%) mutant lines (Table S4).Homozygous mutant lines of gum were analyzed at the bicellular and tricellular pollen stages.It has been demonstrated that GUM affects the MGU association only at tricellular stages (Lalanne & Twell, 2002); therefore, developmental data of the bicellular pollen stages are not shown.This demonstrates that GUM may regulate the cytoskeleton or cellular network which keeps the two components of the MGU close to each other but not the organization and physical assembly of the MGU.

| Presence of physical contact between a SC and VN in gum 1-1
The MGU association has also been analyzed in the second allele of GUM ( gum1-1 À/À ) by introducing different fluorescent markers to label both germline and vegetative nuclei as mentioned in the case of gum1-2.The developmental analysis of five independent homozygous backed-crossed-6 (BC-6) double marker lines of gum1-1 À/À (TET11-GFP Â LAT52:RanGAP-tdTomato; DUO1:TET11-tdTomato Â LAT52:RanGAP-GFP; TET11-GFP Â DUO3:H2B-tdTomato) determines that one SC of pair is physically linked and the MGU association has not been lost in gum1-1 À/À , irrespective that the two SCs are situated far from the VN and the MGU association remains intact (Figure S14).The physical association between germline and vegetative nuclear envelope has been observed through CP at various bud stages in gum1-1 À/À (100%) (Table S5).This determines that the physical association between germline and VN in the form of MGU is completely independent of the proposed GUM regulator and genetic and molecular mechanisms need to be explored.

| DISCUSSION
Morphogenesis and elongation of the GC body and its CP are unique features of angiosperms.However, both developmental processes are regulated independently based on the analysis of multiple mutant alleles of DUO1 (Rauf et al., 2023).In that paper, we have shown that the male gametophyte bicellular pollen mutants (duo1-1, duo1-2, duo1-3, duo1-4, cdka;1, fbl17)/(daz1 daz2, duo2, duo3- Rauf, 2017) form the CP, while here we confirmed the presence of MGU association, which exclude their potential role in this assemblage.The GC possesses CP in wild type (Figure 1 The important role of male germline CP has been reviewed (Dumas et al., 1998;McCue et al., 2011;Mogensen, 1992).The physical linkage of one SC of the pair with the VN, via a CP was reported in cotton pollen grain (Jensen & Fisher, 1968).This association forms the structural and functional unit of the organization, known as the MGU (Dumas et al., 1985).This physical association between the male germline and the VN has also been observed at different bicellular (Figure 1(a,b), Movie S1-S4) and tricellular (Figure 2, Movies S5-S7) developmental stages in A. thaliana and the same in other plant species including Cymbidium goeringii (Yu & Russell, 1992), Tropaeolum majus (Niu et al., 1999), Plumbago zeylanica (Russell & Strout, 2005), and Z. mays (Kliwer & Dresselhaus, 2010).The analysis of fluorescently labeled male gametophytes could help to determine the presence of the MGU in the remaining different species of angiosperm.
The majority of the angiosperms have MGU association (Tables 1 and   2), while our current developmental analysis has demonstrated the striking importance of in vivo imaging for the confirmation of narrow CP of male germline (GC/SC) and the broad conservation of the persistent MGU contact in all the angiosperms and its important role in successful double fertilization.
While the mechanisms regulating ontogeny of the CP and its role in the establishment of the MGU are unknown, it was hypothesized that the physical association between the SC and VN might be disturbed or lost in the gum mutants ( gum1-1/gum1-2) (Lalanne & Twell, 2002; Figure 4).The in vivo developmental analysis of double marker lines of gum1-1/gum1-2 mutants revealed that the physical association between the SC and VN is sufficient to prevent dissociation, despite separation of the two structures in gum (Figures 4 and 5).
We have analyzed five independent homozygous backcrossed-6 (BC6) gum1-1/gum1-2 double fluorescent marker lines, while an intact physical association was observed in the displaced MGU.The pollen development was also analyzed at different bicellular pollen stages in the homozygous backcrossed-6 individuals, but the gum phonotype appeared only at the tricellular pollen stages (Lalanne & Twell, 2002); therefore, data is not shown.Further, it was speculated that bicellular pollen mutants might also show defects in the MGU organization and development of the MGU but an intact association has been observed in pollen of the various mutant alleles including duo1-2, duo1-4, duo2, duo3, daz1 daz2, fbl17, and cdka;1, and thus, the process is independently regulated of these bicellular pollen mutants (Figure 3; Figure S8-S12/S15).

|
The MGU association is unaffected and independently regulated of GUM in A. thaliana The MGU is the structural and functional unit in the majority of angiosperms (Dumas et al., 1984), and an attempt was made to find the genetic and molecular mechanisms of the MGU association and establishment in A. thaliana (Lalanne & Twell, 2002).The in vivo fluorescence microscopic analysis of double marker lines demonstrated an intact MGU in gum (Figure 4/S13-S14), which had been reported might be a potential regulator of the MGU organization and ontogeny in A. thaliana due to the apparent loss of physical linkage (Lalanne & Twell, 2002).Therefore, it remains unresolved to determine which genes/factors regulate the establishment of the MGU in A. thaliana.
The connection between SC and VC nucleus through CP is firm enough that even displacement of the two structures from each other does not cause the dissociation of the linkage in gum mutants (Figures 4 and 5).This provides an opportunity to explore and determine the genetic and molecular mechanisms regulating the establishment of the MGU using A. thaliana.
The MGU association has not been analyzed at early stages in the pollen grain in many plants, and detailed data is limited, for evidence of the GC physical linkage with the VN at bicellular pollen stages (Early MGU) (Kliwer & Dresselhaus, 2010;Palevitz, 1993;Yu & Russell, 1992, 1994) S2).The live pollen analysis using both light and fluorescence microscopy demonstrates that the round GC at early stages positions very close to the vegetative nuclear membrane and appears to be physically linked with the VC nuclear membrane through its CP to form the MGU (Figure 1(a,b), Movie S1).
In Cymbidium goeringii, some of the GCs lose their CPs (Yu & Russell, 1992), or in N. tabacum, the GC extensions are fully or partially withdrawn, and the MGU dissociates at GC division at metaphase and the association is re-established at tricellular pollen stage (Kliwer & Dresselhaus, 2010;Niu et al., 1999;Palevitz, 1993;Yu & Russell, 1994).In contrast, no such loss or shortening of CPs nor dissociation of the MGU was observed at any stage of development in A. thaliana (Figure 1(b); Figure S7; Movies S2-S4).Generally, it is presumed that the male germline body (GC/SC) elongation might be necessary for the association and establishment of the MGU.Our analysis has well established that only CP elongation and development are required for the MGU association since if the GC body undergoes complete (fbl17/cdka;1), partial (duo2/duo3/daz1daz2), or no (duo1) morphogenesis in these different bicellular pollen mutants, an intact MGU can be formed (Figure 2; Figure S8-S10/S15).This further strengthens the discovery that the ontogeny of CP and GC elongation is independently regulated (Rauf et al., 2023).

| The newly formed SCs are physically linked in Arabidopsis
The twin SCs are physically linked with each other, while one of them is physically associated with the VC nucleus via a CP in most tricellular pollen species and bicellular pollen species as mentioned earlier (Tables 1 and 2).We observed a similar pattern at all developmental stages in the male gametophyte developmental analysis in A. thaliana.
Except for a few plant species like H. foetidus that lack the MGU (Heslop-Harrison et al., 1986), where the reported GC appears to have tapering ends (CPs) and the revisiting might confirm the presence of MGU.The MGU is observed in mature pollen at anthesis (Charzyńska et al., 1988) and during the passage of SCs within pollen tubes in H. vulgare (Mogensen & Wagner, 1987), which has been previously reported to show deviation from the rest of Angiosperms during pollen development (Cass, 1973;Cass & Karas, 1975;Mogensen & Rusche, 1985).However, further detailed observations of live cells including MT-labelling are required to confirm the MGU in these species because the fine CP is difficult to visualize without labelling either with fluorescent markers or anti-MT or 3-D reconstruction.The 3-D reconstruction of the MGU in different plant species has shown that the GC is physically associated with a VN through CPs (Theunis et al., 1985;Mogensen, 1986;McConchie, Hough, & Knox, 1987;Zhu et al., 1990;Yu & Russell, 1992;Palevitz, 1993;Yu & Russell, 1994;Russell & Strout, 2005, Hirano & Hoshino, 2010;Reviewed in McCue et al., 2011) and the same was observed in Arabidopsis 3-D analysis (Movies 1-7).Further, in vivo analysis of fluorescent marker lines in tobacco and maize also revealed that the GC CP is linked with the VC nucleus (Kliwer & Dresselhaus, 2010;Oh et al., 2010), while the same was observed in the developmental analysis of A. thaliana (Figures 1-5).
[a,b]; FigureS1-S3a).The GC is round in profile and has a fine thread-like CP (Figure1(b), Movie S1; FigureS1-S3), which extends toward the VC nucleus and is physically associated with the vegetative nuclear membrane.We used various fluorescent markers to examine during the in vivo developmental analysis using this association between the GC CP and the VC nucleus is observed at early, mid, and late bicellular bud stages (Early MGU) and the analysis demonstrated that the GC is physically linked with VC membrane through CP (Figure1(a,b], Movie S2; Figure S1-S3).The length of the CP increases with GC body morphogenesis, and an elongated CP has been observed at the late bicellular stage (Figure 1(b), Movies S3 and S4; Figure S2-S3).The physical association between dividing GC and vegetative nuclear membrane appears to remain intact based on the double fluorescent markers analysis (HTR10:GFP-TUA6 Â DUO3: H2B-tdTomato; HTR10:GFP-TUA6 Â LAT52:RanGAP-tdTomato).The MGU association has been observed using different sets of fluorescent markers, and the same set of data has been obtained at all these bicellular bud stages (Figure 1(a,b); Figure S1-S3; Movies S1-S4).Multiple and different sets of fluorescent markers were used in this study to exclude any artifact and reconfirm the MGU between GC/SC (TET11-GFP/HTR10:GFP-TUA6/DUO1-TET11-td-Tomato) and VN (LAT52:RanGAP-GFP/DUO3:H2B-tdTomato).To visualize the MGU association, it was mandatory to label the male germline (GC/SC) and VN (DUO3:H2B-tdTomato) or vegetative nuclear membrane (LAT52:RanGAP-GFP) with contrast fluorescent tags.Furthermore, HTR10 is a target of DUO1; therefore, the fluorescent marker (HTR10:GFP-TUA6) has no expression in multiple mutant alleles of DUO1 (duo1-1, duo1-2, duo1-3, duo1-4),
, N. tabacum F I G U R E 6 Schematic development of pollen showing MGU association at the bicellular pollen and tricellular pollen stages.The polarized haploid microspore (a) divides asymmetrically (PMI) into bicellular pollen with a lens-shaped generative cell (GC) attached to the pollen wall (b).The round generative cell is positioned close to the vegetative nucleus (VN), while a physical link through a cytoplasmic projection is established between the generative cell and vegetative nucleus (i.e.early MGU-c-d).The elongated generative cell has a long cytoplasmic projection, whereas the latter links with the vegetative nucleus (e-f).Two sperm cells are formed through symmetric division (PMII) and one of the sperm cells has a physical linkage with the vegetative nucleus (late MGU-g).The black dots represent lipid droplets, which have a high concentration on the surfaces of the plasma membrane (GC/SC).PMI, pollen mitosis-I; PMII, pollen mitosis-II.

(
. It has also been determined during in vivo developmental analysis that the round GC is situated very close to the VN and linked with the vegetative nuclear membrane through CP and forms the MGU at different bicellular (Early MGU) (Figure 1(a,b), Movies S1-S4) and tricellular (Late MGU) (Figure 2, Movies S5-S7) bud stages in A. thaliana (Figure 6), while a recent review proposed the presence of MGU association only at late bicellular and tricellular pollen stages (McCue et al., 2011).Most of the previous developmental analyses have been performed to analyze the MGU association between the GC CP and VN using either immunolocalization of MT or electron microscopy or 3-D reconstruction.In the present study, various alternate strategies were used to mark the GC CP (cytoplasm/ microtubule marker-HTR10:GFP-TUA6; plasma membrane marker-TET11-GFP/DUO1:TET11-tdTomato), the VC nuclear membrane (LAT52:RanGAP-tdTomato), and both GC and VC nuclei (DUO3:H2B-tdTomato) (Table GCs and SCs develop fine CPs, and thus, all angiosperms potentially have the MGU association during male gametophyte development based on our analysis.Therefore, it seems that there might be an intact association between male germline cells and VN throughout pollen development in flowering plants based on the in vivo developmental analysis of multiple fluorescent marker lines of A. thaliana as well as a review of the published literature of various plant species.The molecular and genetic mechanisms of the CP's ontogeny and MGU association are still unknown.The developmental analysis of different pollen mutants demonstrates that GC cytoplasmic extension ontogeny and MGU association are not controlled by DUO1, DAZ1, DUO3, DAZ1 DAZ2, DUO2, FBL17, CDKA;1, and GUM in A. thaliana.Based on the available data from different pollen mutants, it seems interesting that the initiation of CP and the MGU association might be regulated by the same genes or pathways during microgametogenesis in angiosperms.To unfold this mystery, we need to a male gametophyte mutant without CP and then developmental analysis for the existence of a MGU.If a MGU is still established in the absence of a CP, then both are independently regulated, otherwise not.Therefore, gametophytic screening of mutagenized marker lines may be a promising way forward to identify the structural and regulatory components that determine MGU assembly and CP development in future work.AUTHOR CONTRIBUTIONS Experiments were conceived by AR and DT and executed by AR.The manuscript was written by AR and reviewed by JL and DT.The literature survey of MGU's existence was performed in BCP (AW, YL, ZL, MW) and TCP (IK, KJ, SW, MK).